5 Current Address: Clinical Research Program, Division of Allergy, Immunology and Transplantation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA

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Abstract

Background

In 2009, xenotropic murine leukemia virus-related virus (XMRV) was reported in 67%
of patients with chronic fatigue syndrome (CFS) compared to 4% of controls. Since
then numerous reports failed to detect XMRV in other cohorts of CFS patients, and
some studies suggested that XMRV sequences in human samples might be due to contamination
of these samples with mouse DNA.

Results

We determined the prevalence of XMRV in patients with CFS from similar areas in the
United States as the original 2009 study, along with patients with chronic inflammatory
disorders and healthy persons. Using quantitative PCR, we initially detected very
low level signals for XMRV DNA in 15% of patients with CFS; however, the frequency
of PCR positivity was no different between patients with CFS and controls. Repeated
attempts to isolate PCR products from these reactions were unsuccessful. These findings
were supported by our observations that PHA and IL-2 stimulation of peripheral blood
mononuclear cells from patients with apparently low levels of XMRV, which induced
virus replication in the 2009 report, resulted in the disappearance of the signal
for XMRV DNA in the cells. Immunoprecipitation of XMRV-infected cell lysates using
serum from patients from whom we initially detected low levels of XMRV DNA followed
by immunoblotting with antibodies to XMRV gp70 protein failed to detect antibody in
the patients, although one control had a weak level of reactivity. Diverse murine
leukemia virus (MLV) sequences were obtained by nested PCR with a similar frequency
in CFS patients and controls. Finally, we did not detect XMRV sequences in patients
with several chronic inflammatory disorders including rheumatoid arthritis, Bechet's
disease, and systemic lupus erythematosus.

Conclusions

We found no definitive evidence for XMRV DNA sequences or antibody in our cohort of
CFS patients, which like the original 2009 study, included patients from diverse regions
of the United States. In addition, XMRV was not detected in a cohort of patients with
chronic inflammatory disorders.

Keywords:

Background

Chronic fatigue syndrome (CFS) is characterized by debilitating, unexplained, persistent
or relapsing severe fatigue of new onset that is not relieved by rest or reduction
of activities. In addition, criteria for CFS require that patients concurrently have
four or more of the following symptoms for ≥6 months (a) impaired memory or concentration,
(b) sore throat, (c) tender cervical or axillary lymph nodes, (d), muscle pain, (e)
multi-joint pain without redness or swelling, (f) headache, (g) unrefreshing sleep,
or (h) post-exertional malaise. While a large number of infectious agents have been
postulated to cause CFS, further studies have not confirmed these findings. In 2009,
Lombardi et al. [1] first reported the presence of xenotropic murine leukemia virus-related virus (XMRV)
in the blood of 67% of patients with CFS compared with 3.7% of control subjects. In
a recent study, Lo et al. [2] reported the presence of murine leukemia virus (MLV)-related virus gene sequences
in 86.5% of CFS patients and 6.8% of controls. The sequences amplified by nested PCR
from these patients were distinct from XMRV reported by Lombardi et al. [1]. Recently, a number of other studies have failed to confirm this observation [3-10]. Recent studies have suggested that amplification of XMRV DNA in human samples is
due to contamination of these samples with mouse DNA [11-15].

In view of the controversies linking CFS to MLVs among different laboratories, we
tested our well characterized cohort of chronic fatigue syndrome patients that fulfilled
the CDC case definition [16] for both XMRV and MLV-related viruses. We failed to find definitive evidence for
XMRV DNA sequences or antibody in our cohort of CFS patients, which were from diverse
areas of the United States, similar to the cohort reported in original 2009 study
[1,17]. We did, however, detect a diverse set of MLV-related virus gene sequences at a similar
frequency in CFS patients as in healthy individuals.

Results

A very weak signal is detected for XMRV in PBMCs from some patients with CFS, but
the frequency of PCR positivity is not significantly different from controls

In the first set of experiments, we determined the frequency and level of XMRV DNA
in blood obtained from cohort 1 which included patients with CFS (21-61 years), idiopathic
chronic fatigue, other viral diseases, and healthy blood bank donor controls obtained
from 1993-2007 (Table 1). As reported previously for patients with CFS [7-9], most of the patients and controls in the cohort were Caucasian women ages 40-45.
Most patients and controls were from the Midwest or Southern United States; other
patients were from the Northeastern and Western United States.

Real-time qPCR was performed using primers for a portion of the XMRV integrase gene
[18] by a scientist who was blinded to the identity of the samples. The PCR assay could
reliably detect 5 copies of XMRV DNA per reaction in the presence of 250 ng of cellular
genomic DNA (or 20 copies of XMRV DNA/ug cellular DNA). Positive samples were defined
as those having detectable DNA in the majority of replicates; at least 3 replicates
were performed on each sample. Nine of 61 (15%) samples from patients with CFS were
positive for XMRV DNA in the real-time qPCR assay. The mean DNA copy number of the
positive samples was 21 copies per ug of cellular DNA, with a range of 9.9-37 copies
per microgram DNA. There were no significant differences in the age, gender, or mean
years of illness in XMRV-positive vs. XMRV-negative persons (Table 1). Patients with low levels of XMRV DNA were more likely to be from the Midwest and
less likely to be from the Northeast. In contrast, none of the 18 subjects with idiopathic
chronic fatigue, non-CFS viral disease, or healthy blood bank donors in the cohort
was positive for XMRV. The difference between the PCR positivity for XMRV in patients
with CFS versus patient controls or healthy blood donors in cohort 1 was not significant
(p = 0.593). Therefore, the very weak signal (less than twice the lower limit of reliable
detection) for XMRV in the minority of patients with CFS was not conclusively associated
with this disease.

PCR was performed on patients with CFS that were positive for XMRV DNA using primers
specific for mouse GAPDH or IAP sequences. All of the samples were negative for mouse
GAPDH or IAP DNA; in contrast genomic DNA from mouse cells yielded the predicted size
(140 bp for GAPDH and 300 bp for IAP DNA) band in the assay (data not shown).

We then measured the level of XMRV DNA in the blood from cohort 2 which included 50
healthy persons and 97 patients with chronic inflammatory diseases including 30 with
rheumatoid arthritis, 20 with Behcet's disease, 10 with systemic lupus erythematosus,
and 9 with cryopyrin-associated periodic syndromes (Table 2). All 97 samples were negative for XMRV DNA. To confirm that the genomic DNAs used
did not contain any inhibitor that could account for lack of amplification, the RNAse
P sequences were amplified using an RNAse P qPCR kit; all 226 samples were positive
for RNAse P.

Table 2. Results of PCR for patients with chronic fatigue syndrome and controls.

We then tested DNA from all 226 samples in a real-time qPCR assay for a portion of
the XMRV envelope gene [3]. This PCR assay could not detect fewer than 15 copies of XMRV per 250 ng of genomic
DNA; therefore, it was less sensitive than the XMRV integrase PCR. All 226 DNAs were
XMRV-negative using the XMRV envelope assay. These results are similar to prior results
in which XMRV sequences could not be detected in samples from a CFS cohort from the
United Kingdom [3].

XMRV cannot be detected in PBMCs from patients with CFS by activation of the cells

These data prompted us to carry out further experiments on a subset of qPCR-positive
and negative samples by looking for the amplification of XMRV proviral DNA after activation
of patient PBMC by PHA and further stimulation with IL-2. No amplification was observed
using the integrase based qPCR assay, including those that scored positive in the
initial assay. In addition, repeated attempts to isolate and clone PCR-amplified DNA
from a subset of the qPCR-positive samples were unsuccessful. This suggests that the
samples that were weakly positive initially in the CFS patients may have been due
to non-specific amplification of cellular DNA or a falsely positive signal.

PBMCs from patients with CFS are not more likely to have a signal for MLV-related
viruses than controls

Recently MLV-related virus DNA was detected in patients with CFS using nested PCR
[2]. Initially we used Taq polymerase (Invitrogen) that does not contain the mouse monoclonal
antibody present in Platinum Taq. However, no signal was observed in the patient or
control samples. Therefore, we used Platinum Taq polymerase (the Taq polymerase used
by Lo et al. [2], but the no template controls also amplified a DNA product. However, a new lot of
Platinum Taq polymerase was selected which did not amplify a DNA product in the no
template controls. When this Taq polymerase was used with genomic DNA template in
a nested PCR assay, a 400 bp DNA was readily amplified from both CFS patients and
non-CFS subjects (9 healthy blood donors, 10 patients with rheumatoid arthritis, and
9 other patients). The PCR amplification products from 14 CFS patients and 8 healthy
donors were sequenced and aligned to sequences in Genbank using a BLAST search. We
identified at least 5 distinctive gag sequences homologous to endogenous MLVs of the polytropic (Pmv), modified polytropic
(Mpmv), or xenotropic (Xmv) subgroups (Table 3). The sequence from one control (healthy donor 2) is an MLV-like gag containing a
region highly homologous to Mpmv MLV followed by sequence with lower homology to known
MLVs. Most of these PCR products were also highly related to MLV-related sequences
that were previously identified in samples from patients with CFS or prostate cancer
[2,19,20]. In addition, a portion of human chromosome 1 was amplified from two CFS patients
and one control sample. While the numbers of samples analyzed were small, MLV-related
virus sequences were detected in both patients with CFS and in healthy controls indicating
that the amplification of viral DNA was not specific for CFS. No PCR product was obtained
in the absence of a template or using African green monkey (Vero) cells or telomerase-transformed
rhesus fibroblasts (Telo-RF cells) grown in cell culture.

Patients with CFS and a weak signal for XMRV do not have detectable antibody to XMRV

Since Lombardi et al. [1] detected antibody to XMRV in many of their patients with CFS, we also looked for
antibody in our patients. Immunoprecipitation of XMRV antigens from mock-infected
or XMRV-infected ferret cells using serum from CFS patients and non-CFS controls,
followed by immunoblotting with a polyclonal goat antibody to XMRV gp70 did not reveal
the presence of antibodies to XMRV gp70 antigen (Figure 1). A positive control, in which an XMRV gp70-reactive rat monoclonal antibody 83A25
[21] was used to immunoprecipitate gp70, readily detected gp70 from XMRV-infected ferret
cells (Figure 1A, B last lanes). A faint band co-migrating with gp70 was observed in the serum from one
subject without CFS using either XMRV-infected 293T or ferret cells. The origin of
this faint band was not further investigated. Similar results, showing lack of detectable
antibody to XMRV, were also observed in virus-infected 293T cells (data not shown).

Figure 1.Absence of antibody to XMRV gp70 in serum from patients with CFS. XMRV gp70 protein was immunoprecipitated from XMRV-uninfected (Un) or XMRV-infected
(XMRV) ferret cells using serum from subjects with (CFS) or without (Cont) CFS or
with a rat monoclonal antibody to gp70. Immune complexes were separated on 4-20% SDS
gels transferred to nitrocellulose membranes and immunoblotted with goat polyclonal
antibody to XMRV gp70. (A) Sera from CFS patients 13 and 48, and control subject 22.
(B) Sera from CFS patients 59 and 62 and control subject 87. The numbers between the
blots are sizes of protein markers in kilodaltons.

Discussion

We initially detected very weak signals for XMRV in 15% of patients with CFS with
a set of XMRV primers using real-time PCR, but failed to detect XMRV in patients with
idiopathic chronic fatigue, chronic inflammatory disorders, other viral infections,
or healthy controls. The difference in frequency of a weak PCR positive signal for
XMRV for patients with CFS versus controls was not significant, thus, these results
did not indicate a clear relationship between XMRV and CFS. Our repeated failure to
isolate and clone XMRV sequences by PCR from the samples that were low positive in
the real-time PCR assay, suggests that the low positive signals were false-positive
artifacts, rather than contamination with mouse DNA. Tuke et al. [22] reported that Invitrogen Platinum Taq PCR Master Mix was contaminated with mouse
DNA sequences; however, they did not detect murine sequences in Applied Biosystems
Taq PCR Master Mix. Since we also used Applied Biosystems Taq PCR master mix for amplication
of DNA by real-time PCR, this further supports the likelihood of a false positive
signal as an artifact, rather than contamination with XMRV. We were also unable to
detect XMRV in patients with CFS using a different set of XMRV primers or in PBMCs
activated by PHA and further stimulated with IL-2 to amplify XMRV DNA. These latter
results are in contrast to those of Lombardi et al. who found that activation of PBMCs
from patients with CFS with PHA induced expression of virus proteins and infectious
viral particles [1], which would be expected to amplify XMRV DNA.

Since the original report of Lombardi et al. [1] in which XMRV DNA was detected in 67% of CFS patients and 3.7% of controls, there
are numerous reports from the United Kingdom [3,4], the Netherlands [5], China [6], Japan [12,23], and the United States [7-10,19,24] that did not detect XMRV DNA in patients with CFS. Reasons postulated for the difference
in results include geography of the patients, contamination of samples or reagents
with mouse DNA, sequence variation in XMRV, and definitions of CFS.

The blood samples we evaluated were collected from the NIH cohort of CFS patients
during 1993-1995 and patients were predominantly from Midwestern and Southern United
States, but also included patients from Western and Northeastern United States. Lombardi
et al. studied patients from the 2006 to 2008, including some patients identified
during an outbreak of CFS in 1984-1988, and their patients came from at least 11 states
in various regions of the country [1,17]. Like our cohort, their patients were from diverse areas of the United States. Prior
reports generally have studied more recent patients and most of the patients have
been from more restricted locations in the United States or different geographic areas
than those of the original report. Our study more closely resembles that of Satterfield
et al. [24] who studied patients from multiple states of the continental United States, whereas
other studies have reported patients from a single geographic area of the United States
[8-11], two states [7], or other countries [3-6,23,25].

Using the nested PCR assay reported by Lo et al. [2], we detected MLV-related viral DNA in all human samples tested, regardless of whether
they were from patients with CFS, inflammatory diseases, or normal controls. Sequence
analysis showed that sequences in both CFS and healthy controls aligned with MLV sequences
found in mice and reported in patients with CFS or prostate cancer [2,19,20]. In contrast, we did not detect MLV-related virus in monkey cell lines. While the
nested PCR findings suggest that the human samples were contaminated with mouse DNA,
specific testing for mouse genomic DNA (testing for both mouse GAPDH and multi-copy
mouse IAP transposons) was negative. In addition, nested PCR of monkey cell DNA did
not amplify MLV sequences. Thus, the MLV sequences might be due to contamination during
DNA isolation (from the AllPrep DNA/RNA isolation kit), from Platinum Taq (as reported
by Tuke et al. [22], or due to very low levels of MLV related sequences in human DNA. Using other procedures
for isolating DNA and PCR, Satterfield et al. [24] did not detect MLV in CFS patients from 17 states in the United States.

Our serologic analysis by immunoprecipitation of gp70 from XMRV-infected 293T or ferret
cells using CFS patient sera and sera from non-CFS control patients did not detect
XMRV gp70 specific antibodies in patients with CFS. Our results are consistent with
those of Erlwein et al. [25], Satterfield et al. [24], Knox et al. [9] and Shin et al. [10] who were unable to detect antibody to XMRV in patients with CFS.

Since this work was performed, Paprotka et al [15] and Knox et al [9] have shown that XMRV originated sometime between 1993 and 1996 from recombination
between two endogenous MLVs during tumor passaging in mice, and that XMRV could not
be detected in 43 patients who had previously been reported XMRV positive. Our findings,
indicating no definitive evidence linking XMRV with CFS both by PCR and by antibody
testing, support those of others that XMRV is not a cause of CFS. Thus, the search
must continue for other etiologies for CFS.

Conclusions

We did not find evidence linking XMRV to CFS by PCR of PBMCs or by immunoblotting
of patient serum. Furthermore we did not find XMRV sequences in patients with connective
tissue disorders including rheumatoid arthritis, Bechet's disease, and systemic lupus
erythematosus.

Methods

Patients, controls, and DNA isolation

Subjects in cohort 1 with CFS, idiopathic chronic fatigue, non-CFS viral disease,
and healthy blood donors were obtained from protocols approved by the Institutional
Review Board (IRB) of the National Institute of Allergy and Infectious Diseases (NIAID).
All patients signed written consents and blood had been obtained during 1993-1995.
CFS was defined using Centers for Disease Control and Prevention criteria [16]. Idiopathic chronic fatigue was defined as clinically evaluated, unexplained chronic
fatigue that failed to meet criteria for the chronic fatigue syndrome [16]. Healthy donors had signed consents on an NIAID approved protocol.

Since CFS has been associated with chronic immune activation, we tested a second group
of patients, Cohort 2, which included patients with inflammatory diseases who had
signed consents as part of protocols approved by the joint National Institute of Diabetes,
Digestive and Kidney Diseases/National Institute of Arthritis and Musculoskeletal
and Skin Diseases IRB.

Genomic DNA was extracted from 5-10 × 106 peripheral blood mononuclear cells (PBMCs) in patients from cohort 1 that had been
cryopreserved in liquid nitrogen using an AllPrep DNA/RNA isolation kit (Qiagen, USA).
DNA from PBMCs in patients from cohort 2 was extracted using various procedures.

PHA-induction and IL-2 stimulation of PBMCs

PBMCs from a subset of cohort 1 were activated by 0.5 ug/ml of PHA-L (Roche Diagnostics),
and after 72 hours, cells were cultured with 20 U/ml of recombinant IL-2 (Biological
Resource Branch, NCI) and subcultured every 3-5 days as described earlier [1]. Genomic DNA was prepared on day 8 from IL-2-stimulated cells using an AllPrep DNA/RNA
isolation kit as described above.

Real-time quantitative PCR of XMRV DNA

A 121 bp region from the XMRV integrase gene was amplified by real-time quantitative
PCR (qPCR) using primers 4552F and 4673R and a 5'FAM and 3'TAMRA labeled probe 4572
as described previously [18]. A second real-time qPCR assay for a 71 bp fragment from the XMRV envelope gene used
primers 6173 env F, 6173 env R, and 5'-FAM and 3'-TAMRA labeled 6173 envelope probe
as described previously [3].

Reaction mixtures for qPCR contained 1X TaqMan Universal PCR Master Mix (Applied Biosystems),
1 uM each of primer, 250 nM of probe and 250 ng of genomic DNA in a total volume of
25 ul. All PCR reactions were performed with duplicate samples. Reactions were performed
using an ABI 7500 real-Time PCR System (Applied Biosystems) with the following conditions:
50°C for 2 min, 95°C for 10 min, 40 cycles at 95°C for 20 sec and 60°C for 1 min.
A standard curve consisting of 10-fold serial dilutions (5-50,000 copies) of XMRV
proviral DNA VP62 [26; a gift from F. Ruscetti, NCI] spiked with 250 ng of denatured
salmon sperm DNA was amplified using identical conditions to quantify the presence
of XMRV DNA in the human samples. The assay can reliably detect 20 copies of XMRV
DNA/ug of cellular DNA. To ensure that no inhibitor was present, the single copy RNAse
P gene was amplified from each 250 ng DNA sample using a TaqMan RNAse P Detection
Reagent kit (Applied Biosystems).

PCR amplification for XMRV and MLV-related virus DNA

Nested PCR of MLV-related virus and XMRV gag sequences was performed as described
by Lo et al. [2]. XMRV DNA was amplified for 40 cycles using primers 419F and 1154R with 50 ng of
genomic DNA and 1 U of Platinum Taq polymerase (Invitrogen) followed by 45 cycles
in the 2nd round using either primers GAG-1F and GAG-1R for XMRV DNA, or primers N116 and N117
for MLV-related virus [2]. Multiple controls without template were run in each PCR experiment.

PCR amplification of mouse DNA

The presence of mouse DNA was tested by PCR for mouse GAPDH which is present at 2
copies/mouse genome as described earlier [26]. Low level contamination of mouse DNA was tested by amplification of intracisternal
A particle (IAP) transposons which are present at ~1000 copies/mouse genome as described
previously [11]. Serial dilutions of mouse DNA were used as positive controls, and PCR products were
analyzed by electrophoresis on agarose gels.

Immunoprecipitation and immunoblotting for XMRV gp70 protein

The presence of antibodies to XMRV in human serum samples was tested by immunoprecipitating
XMRV antigens from XMRV-infected or mock infected 293T or ferret cell extracts. Ferret
cells were found to yield a higher level of infection than human cell lines (e.g.
HeLa, 293T cells). 293T cells were obtained from the American Type Culture Collection
(Manassas, VA) and MA-139 ferret cells [27] were a gift from Janet Hartley, NIAID, Bethesda, MD. Briefly, about 40 ul of cell
extracts were mixed with 280 ul of 1× RIPA buffer (10 mM Tris-HCl, pH 8.0, 150 mM
NaCl, 1 mM EDTA, 1.0% NP-40, 0.5% deoxycholate and 0.1%SDS). Human serum (20 ul) or
cell culture filtrate containing rat monoclonal antibody 83A25 [19] to XMRV gp70 (20 ul) was added. The mixture was incubated on ice for 1 h. Then 40
ul of a 50% suspension of protein-G-Sepharose (Sigma-Aldrich) was added and the antigen-antibody
reaction was allowed to proceed for 12-16 h at 4°C in a rotator. Immune complexes
were purified by washing the beads 3-4 times with 1× RIPA buffer and boiled for 3
min in 1× SDS-PAGE loading buffer (Quality Biologicals). Proteins were separated on
4-20% Tris-glycine SDS-PAGE gels (Invitrogen), transferred to nitrocellulose membranes,
and incubated with a goat polyclonal antibody to XMRV gp70 (produced by Viromed Biosafety
Laboratories, Camden NJ) at a dilution of 1:3000. After washing, the membrane was
incubated with HRP-conjugated donkey anti-goat antibody (Santa Cruz Biotechnology).

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

All authors read and approved the final manuscript. MAA performed the experiments
and wrote the paper. JKD and SES conducted clinical research and contributed patient
samples and provided demographic data. CAK provided reagents for XMRV and advice on
XMRV. RGM and FWM provided control samples. JIC designed the experiments and wrote
the paper.

Acknowledgements

This work was supported by the Intramural Research Programs of the National Institute
of Allergy and Infectious Diseases, the National Institute of Arthritis and Musculoskeletal
and Skin Diseases, and the National Institute of Environmental Health Sciences. We
thank Francis Ruscetti for XMRV VP62 plasmid and Jing Qin for performing statistics
and Terrance O'Hanlon for technical assistance.